Field of the Invention
Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite which infects a wide variety of animals. This invention relates to novel primers which can be utilized in a polymerase chain reaction (PCR) assay to detect and quantitate the presence of T. gondii in test samples.
Description of the Related Art
T. gondii can exist in all mammals and is believed to persist for the life of its host as cysts in muscle tissue or in the central nervous system. The cysts may rupture, releasing tachyzoites, which are the highly infective stage of the parasite and responsible for the onset of toxoplasmosis, a disease which is potentially fatal in individuals having compromised immune systems. Tachyzoites differentiate into bradyzoites which are located within the cysts and may convert back into the infectious tachyzoite stage should the immune system of the host fail or become compromised.
Toxoplasmosis is one of the most prevalent parasitic diseases in human beings and agricultural animals. Mead et al. (1999. Emerg. Infect. Dis. vol. 5, pp. 605-625) have estimated that approximately 225,000 new cases are reported each year in the U.S., and 50% of the cases are believed to be due to the food borne transmission of T. gondii. The National Hospital Discharge survey indicated that toxoplasmosis was the first diagnosis for approximately 5,000 discharges each year during 1992 to 1996, including 750 deceased patients. Furthermore, 4,000 persons with AIDS will develop toxoplasmic encephalitis in the U.S. each year (Mead et al., supra). In addition, toxoplasmosis may occur during pregnancy in mothers who have not previously been infected with the parasite. From an economic point of view, T. gondii infection has a negative impact on society as measured by increases in the costs of chemotherapy in AIDS patients (Freedberg et al. 1998. J. Am. Med. Assoc. vol. 279, pp. 130-136), serological screening in pregnant women, patient care, loss of productivity and the treatment of infected mothers and children (Lappalainen et al. 1995. Scan. J. Infect. Dis. vol. 27, pp. 265-272).
T. gondii can be transmitted to humans by ingesting T. gondii oocysts in food or water or by consuming tissue cysts in raw or undercooked meat. Infected pork is considered the most important meat source of T. gondii in the U.S. (Dubey, J. P. 1994. J. Am. Vet. Med. Assoc. vol. 205. pp. 1593-15984). Considering the potentially serious consequences of congenital T. gondii infection in humans, such as birth defects, retinitis, brain damage, or even death, it is essential that efforts be directed at preventing food borne transmission.
The detection of T. gondii cysts in tissues of naturally and experimentally infected pigs and sheep has been reported for years (Dubey et al. 1986. J. Am. Vet. Med. Assoc. vol. 188, pp. 1035-1037; Jacobs et al. 1960. J. Parasitol. vol. 46, pp. 23-28), although the prevalence of T. gondii infection in U.S. pig populations has been dramatically reduced as producers have modified their management practices (Weigel et al. 1995. J. Parasitol. vol. 81, pp. 736-741). A true T. gondii burden in any food product has been difficult to measure, however, and to date, the most reliable method of food inspection for T. gondii has been to demonstrate the presence of T. gondii tissue cysts by in vivo biological assays (Esteban-Redondo et al. 1999. Vet. Parasitol. vol. 86, pp. 155-171; Gamble and Murrell. 1998. Parasitology. vol. 117, pp. S97-111). Because these methods are costly, time-consuming and often unreliable, they are not suitable for slaughterhouse testing or for monitoring commercial meat products (Gamble and Murrell, supra).
There have been many reports of molecular-based assays for the diagnosis of toxoplasmosis, but most have been developed for human diagnostics (Costa et al. 2000. J. Clin. Microbiol. vol. 38, pp. 2929-2932;Dupon et al. 1995. J. Clin. Microbiol. vol. 33, pp. 2421-2426; Jenum et al. 1998. Acta Pathol. Microbiol. Immunol. Scand. vol. 106, pp. 680-686) or phylogenetic studies (Carreno and Barta. 1999. J. Parasitol. vol. 85, pp. 77-83; Ellis et al. 1998. Mol. Cell. Probes. vol. 12, pp. 1-13; Kaufman et al. 1996. Mol. Cell. Probes. vol. 10, pp. 289-297). In veterinary medicine, tests for detection of T. gondii by PCR were reported mostly for companion animals (Lappin et al. 1996. Am. J. Vet. Res. vol. 57, pp. 1589-1593; Stiles et al. 1996. Am. J. Vet. Res. vol. 57, pp. 264-267) or sheep (Esteban-Redondo and Innes. 1998. Int. J. Parasitol. vol. 28, pp. 1459-1466; Owen et al. 1998. Vet. Rec. vol. 142, pp. 445-448), though Warnekulasuriya et al. (1998. Int. J. Food Microbiol. vol. 45, pp. 211-215) reported using PCR to identify T. gondii in cured meat products. Several studies have been directed at quantitating the actual T. gondii burden in biological fluids or tissues, but these involved time-consuming PCR protocols (competitive PCR) followed by agarose gel image analysis (Homan et al. 2000. Int. J. Parasitol. vol. 30, pp. 69-75; Kirisits et al. 2000. Int. J. Parasitol. vol. 30, pp. 149-155; Luo et al. 1997. J. Parasitol. vol. 83, pp. 1070-1074). Costa et al. (supra) addressed the quantitation issue with a real-time PCR analysis of T. gondii in human serum samples from stem cell-transplanted patients; however, this assay was based on the T. gondii B1 gene and not the more abundant rRNA gene.
We have designed novel oligonucleotide sequences which are capable of uniquely amplifying DNA from T. gondii when utilized in a PCR assay. In addition, a probe has also been designed which is effective in PCR assays for the detection of T. gondii amplification products. The novel probe is particularly effective when utilized as a fluorogenic probe in a real time PCR assay.
Accordingly, it is an object of the invention to provide novel oligonucleotides for use as primers in a PCR assay for the detection and/or quantitation of T. gondii. 
It is another object of the invention to provide a novel probe for use with the novel primers for the detection and/or quantitation of T. gondii, preferably a fluorogenic probe for use in a fluorogenic real time PCR assay.
It is a further object of the invention to provide PCR assay methods for the detection of T. gondii utilizing the novel primers.
Other objects and advantages of the invention will become readily apparent from the following description.